042
Anal. Chem. 1907, 59,842-846
Thin-Layer Square Wave Voltammetry and Square Wave Stripping Voltammetry Vinay Kumar* Physical Sciences Department, Northern Kentucky University, Highland Heights, Kentucky 41076 William R. Heineman Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221
The feaslbllity of combinlng the highly sensitive techniques of square wave voltammetry (SWV) and square wave strlpping voltammetry (SWSV) with a commercially available thin-layer electrochemical cell having a single-worklng electrode Is demonstrated. The characteristics of thln-layer SWV were investigated by using ferri-/ferrocyanide and diazepam (Valium) systems. The callbration plot data for dlazepam are linear between 10 and 60 ppm with a detectlon limlt of 0.06 ppm. With a Hg-coated glassy carbon electrode, SWSV studies were carried out on 30-pL aqueous solutions of In3+ and Pb2+ ions. The callbration curve for In3+ Is linear up to 2000 ppb with a detectlon limit of 8 ppb. The detectlon limit for lead Is 11 ppb.
The analytical sensitivity of differential pulse voltammetry (DPV) and the small volume capability of the thin-layer electrochemical cell have been combined for the determination of small amounts of analyte (1-3). Heineman et al. have utilized thin-layer differential pulse anodic stripping voltammetry (DPASV) for the determination of Pb2+,Cd2+,Zn2+, Cu2+,and T1+ in the ca. 5 X to 4 x lo4 M range on 70-pL samples (3.5-280 pmol) (2, 2). Thin-layer DPV has been demonstrated with the determination of drugs diazepam (2) and chlorpromazine (3)in the ca. 5 X to 2 X M range on 25-pL samples (1.3-5000 pmol). Recently, a flow system for the trace quantitation of In3+and Pb2+by ASV using linear potential sweep or differential pulse waveforms has been described by Wise and Heineman (4). A detection limit of 2 X 10-l' M was achieved by deposition from a 1-mL sample (0.2 pmol). The theory and experimental verification for the application of a square wave waveform to a reversible redox system under conditions of semiinfinite diffusion have been reported (5-8). In square wave voltammetry (SWV) good detection limits are achieved through efficient discrimination between the charging and the faradaic currents (7). Large scan rates (2500 mV/s) enable rapid acquisition of voltammograms. In addition, this feature facilitates fast repetitive scanning with signal averaging, thus permitting improved signal-to-noise ratio. When SWV is coupled with the anodic stripping technique, higher sensitivities permit shorter deposition times. Using a static mercury drop electrode and a mini flow-through cell, Buchanan and Soleta have carried out automated square wave anodic stripping voltammetry studies on lead and cadmium (9, 10) This paper describes results that demonstrate the feasibility of combining the techniques of SWV and square wave stripping voltammetry (SWSV) with a commercially available LCEC thin-layer cell. In our work, we have utilized the square wave voltammetric waveform that combines a large-amplitude square wave modulation with a staircase waveform (22). This technique. reported by Osteryoung and Osteryoung ( I 2 1 ,
differs from that of Barker, in which a small-amplitude square wave modulation is imposed on a slowly varying DC potential ramp. The small volume (- 10 pL) of the thin-layer cell used in this study permits handling of extremely small samples. The characteristics of thin-layer SWV were investigated by using ferri-/ferrocyanide and the drug diazepam as examples of reversible and irreversible systems, respectively. Thin-layer SWSV was evaluated on Pb2+and In3+as representative analytes.
EXPERIMENTAL SECTION Apparatus. A BAS-100 Electrochemical Analyzer (Bioanalytical Systems, West Lafayette, IN) that is capable of performing the faster SWV and SWSV techniques was used. The voltammograms were plotted on a HIPLOT digital plotter (Houston Instrument). A BAS thin-layer flow cell with a single-working electrode was used in the stationary mode. Both halves of the cell were made of Kel-F. The working electrode portion (lower half) of the cell contained a glassy carbon electrode (3.2-mm diameter) along with the inlet and outlet ports. Two red Teflon gaskets (BAS) of 0.127 mm thickness each were placed between the two halves of the cell, giving a total cell volume of approximately 10 pL. The Ag/AgCl (reference) and the platinum wire (auxiliary) electrodes were both positioned in one of the ports. The other port was used for the syringe injection of the sample or blank into the cell. Reagents. Standard solutions were prepared by diluting 1000 ppm atomic absorption standards (Aldrich for indium and Fisher for lead and mercury) with appropriate volumes of supporting electrolytes. The following supporting electrolytes were used (a) a preelectrolyzed solution of 1 M potassium acetate (J. T. Baker) buffered to pH 4.0 by the addition of glacial acetic acid; (b) 1 M phosphate buffer, pH 7.0, prepared from KH2P04and K2HP0,, and (c) a 2 M solution of KC1. All solutions were prepared with Type I water from a Nanopure Water System (Barnstead-Sybron). A working standard of 2.0 X M K4Fe(CN)6(J.T. Baker) was prepared by diluting a stock solution of the reagent with the KCI stock solution. Procedures. Glassware Cleaning. To remove metal contaminants, all glassware was washed thoroughly with soap and water and rinsed with distilled and nanopure water. Residual organic material was removed by overnight soaking of the glassware in 10% HNO, followed by several rinses with distilled and nanopure water. Glassy Carbon Electrode (GCE)Preparation. For both SWV and SWSV studies the GCE was polished with 0.05-pm alumina (BAS PK-1 polishing kit) on Microcloth (Buehler Ltd., Evanston, IL) according to the instructions supplied with the polishing kit. Before using, each electrode was rinsed well with nanopure water and acetone. In Situ Formed Mercury Film. For SWSV studies the solution to be analyzed was made 20 ppm in Hg2+by the addition of the appropriate amount of 1000 ppm Hg2+ atomic absorption standard. This procedure allows the metal to be determined and the mercury film to be deposited simultaneously on the GCE. At the end of each determination, the potential was stepped to 0.40 V vs. Ag/AgCl and held for 30 s to remove the mercury film. SWV Studies with K4Fe(CN),. The GCE was polished before each set of experiments. The cell was rinsed repeatedly with
0003-2700/87/0359-0842$01.50/0!C 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59,NO. 6,MARCH 15, 1987
843
Table I. Effect of Square Wave Amplitude on Peak Current (Oxidation) and Peak Width at Half-Height' E,, mV, vs.
t o 600
*o
0
-0.300
E(V) vs. Ag/AgCI Figure 1. Thin-layer SW voltammogramof nondeoxygenated2 X lo4 M K,Fe(CN), in 2 M KCI: initial potential = -0.343 V; final potential = +0.600 V; E,, = 25 mV; frequency = 15 Hz; AEs = 4 mV; peak potential = 0.297V vs. Ag/AgCI.
nanopure water before each run. This was accomplished by placing the tip of a microsyringe in one of the ports of the thinlayer cell and pushing the plunger gently. The same procedure was used to fill the cell and both its ports with the nondeoxygenated 2 X lo4 M K4Fe(CN)6solution in 2 M KCI. The solution in the outlet port was necessary for the immersion of the reference and the auxiliary electrodes. Square wave voltammograms were obtained by scanning the potential positively. The normal settings for the parameters used were as follows: initial potential = 0 mV, final potential = 600 mV, square wave amplitude Esw = 25 mV (or higher when effect of square wave amplitude on the peak current was being studied), = 4 mV (or 1mV frequency = 15 Hz, potential step height (AE,) when E,, = 1 mV), quiet time = 10 s, and sensitivity = lod pA/V. At 4-mV step height the scan rate is 60 mV/s and a scan of 600 mV is completed in 10 s. SWSV Studies of Pb2+ and In3+. Electrode polishing, cell cleaning, and filling procedures were the same as described above for the SWV studies. Square wave stripping voltammograms for calibration standards were obtained by applying the appropriate negative potential to the working electrode for 60 s to reduce all of the ions of interest, along with the mercuric ions, to a metal amalgam on the electrode surface. Since dissolved oxygen can cause chemical stripping of the reduced metal, the potential must be negative enough (at least -0.7 V vs. Ag/AgCl) to reduce O2 to HzO and thereby deoxygenate the cell. The supporting electrolyte for both Pb2+and In3+was acetate buffer, pH 4.0. In either case a deposition potential of 4.950 V w. AgfAgCl was used. The voltammograms were recorded by scanning the potential in the positive direction (Ei= -1200 mV and Ef = -250 mV w. AgfAgCl) to strip the metal ion out of the amalgam. The other parameters used were as follows: Esw = 25 mV, frequency = 15 Hz, AE, = 4 mV, scan rate = 60 mV/s, and quiet time = 10 s. Diazepam. A 100-ppm stock solution of diazepam hydrochloride was prepared by dissolving an appropriate amount of the material in 0.1 M HC1. To obtain calibration standards (4-20 ppm), appropriate volumes of the stock solution were diluted with pH 7.0 phosphate buffer. Before the voltammogram was recorded, a potential of -0.800 V was applied for 60 s to remove dissolved oxygen. SW voltammograms were obtained by scanning in the potential range: -500 to -1400 mV vs. Ag/AgCI, the other parameters being the same as described under the procedure for K4Fe(CN)G.Between samples, the cell was flushed with 0.1 M HCl, nanopure water, and then phosphate buffer to minimize sample carryover. R E S U L T S A N D DISCUSSION Characterization of Thin-Layer S W V w i t h Ferro-/ Ferricyanide. In order to characterize SWV in a thin-layer cell, the ferro-/ferricyanide reaction was chosen as a representative reversible system. The effects of changing the experimental parameters associated with the use of a square wave potential waveform (viz., square wave amplitude, frequency, and step height) were examined by obtaining voltammograms with a newly polished GCE in the thin-layer cell.
ESW,m V
i,, PA
WljZ, mV
Ag/AgCl
5 15 20 25 50 100 200
0.2079
140.9 141.8 145.5 146.7 147.3 174.6 -
298 290 292 294 288 284 -
1.380 1.749 2.473 5.256 11.44 -
Nondeoxygenated 2
X
M KaFe(CNInin 2 M KCl.
Figure 1 is a typical SW voltammogram (EP= 297 mV vs. Ag/AgCl) of a freshly prepared, nondeoxygenated 2 X M K,Fe(CN), solution in 2 M KCl. The shape of the wave is essentially identical with that obtained under conditions of semi-infinite diffusion to the electrode. The diffusion layer is much smaller than the cell thickness, e.g., cell thickness = 0.127 mm X 2 z 0.2 mm, and diffusion layer, 1 (2Dt)1/2. Using approximate values of lo4 cm2/s for D (diffusion coefficient) and 10 s (scan time), 1 is calculated to be -0.045 mm. The above calculations show that SWV measurements were made under semiinfinite diffusion conditions rather than the usual thin-layer conditions in which exhaustive electrolysis occurs. Peak Current us. Square Wave Amplitude. The peak current increases linearly with the square wave amplitude, Esw (Table I). This behavior is in agreement with the theoretical predictions made by Christie et al. (5) for SWV in bulk solution. As in pulse voltammetry, increasing the amplitude also leads to increase in the peak width at half height, W1/2 (Table I). Between Esw = 5 and Esw = 50 mV, W,,, increases very gradually. The use of higher square wave amplitudes (e.g., 100-200 mV) results in extremely broad peaks. At Esw = 200 mV, the resolution is extremely poor, and no peak current or potential values are registered. Since the lowest Esw value tried was slightly greater than AE (viz., 4 mV), no distortion of the peak was observed. Table I also summarizes the peak potentials at the various Esw values. Peak Current us. Frequency. T o study the effect of frequency on the peak current, SW voltammograms were recorded on nondeoxygenated 2 x M K,Fe(CN)6 solutions in 2 M KC1 at a constant step height. In the frequency range 5-500 Hz, a near linear relationship between peak current and f1l2was observed. Our data, collected under semiinfinite diffusion conditions in a thin-layer cell, are in accordance with the bulk solution studies of Wojciechowski et al. (12). At f values of 1000 and 2000 Hz, the increase in peak current was nonlinear. As reported by Turner et al. ( 1 3 ) ,this behavior is probably due to charging currents that are caused by the high frequency of the square wave cycles. Peak Current us. S t e p Height. In order to investigate the effect of step height or scan rate (= f AEJ on the peak current, SW voltammograms were obtained on nondeoxygenated 2 x lo4 M K4Fe(CN)6solutions at constant frequency cf = 15 Hz) and at varying AE,values (1-20 mV). On the basis of our data, the peak current is found to be inversely related to the step height. Our results are not in agreement with the theoretical studies reported by Christie et al. (5)for the dropping mercury electrode in bulk solutions in which they predicted that the step height has little effect on the height of the current peak. A probable cause of the deviation may be the iR drop resulting from the uncompensated resistance in the thin-layer cell. This effect has also been observed previously with thin-layer DPV (3, 4 ) . Thin-Layer SWV of Diazepam. The organic compound diazepam hydrochloride is the active drug in the tranquilizer
=
844
ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987 ~
t
500nA I
4
500nA T
u -0250 -0 700
- 1 100
- 1 500
E(V) vs. Ag/AgCI
Figure 2. Thin-layer SW voltammogram of a norideoxygenated 10 ppm dlazepam hydrochloride and pH 7.0 phosphate buffer: inltial potential = -0.700 V; flnal potential = -1.500 V vs. Ag/AgCI; ,Esw= 25 rnV; = 4 rnV. frequency = 15 Hz;
Table 11. Peak Current (Reduction, ip)and Peak Potential ( E , ) Values for Successive SW Voltammograms of Different Aliquots of Diazepam Standards
E,, mV, vs. concn, ppm 10
aliquot no.
i,, WA
AgIAgC1
1
0.72
2
0.50 0.42
-1040 -1052 -1064 -1036 -1040 -1056 -1060 -1036 -1044 -1064 -1096 -1076
3 20
1 2
3 4
30
1
2 50
1
2
3 -
0.90 0.85 0.55 0.45 1.04 0.93 1.79 1.06
0.93
dispensed under the trademark of Valium. Diazepam and its metabolites have been determined by a number of methods, e.g., DPV (2,14), colorimetry (15), gas chromatography-mass spectrometry (16,17),gas-liquid chromatography (18,19),high performance liquid chromatography (20, 21), enzyme immunoassay (22), radioimmunoassay (23, 24), and thin-layer chromatography with fluorescence detection (25). In this study the utility of square wave voltammetry on the electroactive diazepam was tested by obtaining thin-layer voltammograms with the GCE. Figure 2 is an SW voltammogram showing a well-defined reduction wave (EP= -1.076 V vs. Ag/AgCl) for a 10-ppm solution of diazepam hydrochloride in phosphate buffer, pH 7.0, that was not deoxygenated. The removal of dissolved oxygen by holding the potential a t -0.80 V vs. AgJAgCl for 60 s did not result in any significant advantage. The general shape of the peak remained unchanged and the peak height was practically the same also. On the basis of our work the removal of oxygen may not be necessary for SWSV determinations of some species with thin-layer cells. A similar conclusion was arrived a t by Wojciechowski et al. (12) in the case of SWSV on a SMDE. During the calibration plot studies, as successive voltammograms were run on different aliquots for a given concentration of the diazepam solution, the peak current values were found to decrease slightly and the peak potential values were shifted to slightly more negative values (Table 11). In addition, the voltammograms recorded after 4-5 runs were distorted. These phenomena are attributed to fouling of the glassy carbon electrode, probably by deposition of a film of the reaction product on the electrode surface. Attempts to remove the film by dissolving it in acetone and 1:l ethanolphosphate buffer solution were unsuccessful. To overcome
I
I
-05
I
-10
I
I
-1 200
E(V) vs. Ag/AgCI FI ure 3. Thin-layer SW anodic stripping voltammogram for 500 ppb
Id', 20 ppm Hg2+,and 1 M acetate buffer, pH 4.0: deposition at -0.950 V vs. Ag/AgCI for 60 s;E, = 25 rnV; frequency = 15 Hz; A€s = 4 rnV; quiet time = 10 s; scan rate = 60 mV s-'.
the above problems, the working electrode was polished prior to obtaining the voltammogram for each calibration standard. The plot of peak current as a function of diazepam concentration gave a linear relationship between 10 and 60 ppm (Table IV) when this procedure was followed. Our detection limit of 0.06 ppm (2.20 X lo-' M; 6.63 pmol) is slightly lower than the value of 0.1 ppm reported for differential pulse voltammetry (2,14). The detection limit achieved in thin-layer SWV also compares favorably well with those reported by the following methods: colorimetry, 3 ppm (15);gas chromatography-mass spectrometry, 0.2-1.0 ppm ( I 7); gas chromatography, 0.2 ppm (19);radioimmunoassay, 0.01 ppm (23);and thin-layer chromatography with fluorescence detection, 0.75 ppm (25). In a GC-MS determination of diazepam and N desmethyldiazepam, Foltz (16) reported a detection limit of 1 ppb. Even though thin-layer SWV technique is not so sensitive, it does offer the advantage of faster analysis time resulting from the faster scan rates. For example, it takes only 15 s (at 60 mV/s) to obtain an SW voltammogram vs. about 90 s (at 10 mV/s) for the differential pulse voltammogram. In SWV, even higher scan rates (e.g., 200-500 mV/s) are practical and have been successfully tried (12). Among the other advantages are the absence of requirements for deoxygenation, and the convenience of using a commercially available LCEC thin-layer cell. Thin-LayerSWSV of Indium. Indium (In3+)was chosen because of its ideal behavior in stripping voltammetry, the extremely low levels of contamination in water and supporting electrolyte, and our interest in it as a metal label in the voltammetric immunoassay for proteins (26). Well-defined thin-layer SW stripping voltammograms for the oxidation of In deposited in a mercury film electrode were obtained. A representative voltammogram for a 500 ppb In3+ solution in acetate buffer, pH 4, is shown in Figure 3. The peak current for the In oxidation wave was optimized by systematic variation of the square wave amplitude. Figure 4 shows the effect of changing Esw on the peak current for a solution containing 500 ppb In3+ along with 20 ppm Hg2+ in acetate buffer. The peak current was seen to increase gradually as Esw was raised from 10 to 100 mV. However, further increases in Eswresulted in only minor changes in the peak current. This behavior is similar to that obtained for thin-layer DPASV studies (3, 4). Also, as Esw was varied between 10 and 200 mV, an increase in W,,, was noticed. In fact, the use of very large square wave amplitudes (e.g., 100-200 mV) just results in broadening of the peaks, but not causing any increase in their heights. Thus, there is a practical limit to the increase in sensitivity that can be obtained by increasing the square wave amplitude. Our data are in complete agreement with the conclusions arrived at by Turner et
5.0
-
iI
0
1
'
"
"
I
'
'
I
1
/
1
'
'
1
'
'
1
1
0 10 20 30 40 50 60 70 80 90 100110120130140150160170180190200
SWARE WAVE AMPLITUM (mV)
ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987
845
Table V. Effect of Successive in Situ Depositions and Anodic Scans" scan noeb
E,, mV, vs. Ag/AgCl
1 2 3 4
-632 -628 -624 -620
scan no.b
E mV, vs. xgJAgC1
5 6 7
-616 -610 -606
500 ppb In3++ 20 ppm Hg2+in acetate buffer; deposition time = 60 s; Esw = 25 mV; El = -1200 mV; Ei = 100 mV; frequency = 15 Hz; quiet time = 10 s; A E, = 4 mV, deposition potential = -950 mV vs. AdAaCl. Successive scans at 60 V s-l.
F ure 4. Dependence of peak current (oxidation)on ,Esw:500 ppb I%, 20 ppm Hg2+,and pH 4.0 acetate buffer. Other parameters are given in Figure 3.
f
Table 111. Effects of Changing the Square Wave Amplitudea
ESW, mV
200nA
i
i,,b pA
E,, mV, vs. AgJAgCl
W l j z ,mV
0.911 2.10 3.65 4.52 4.53 4.68
-612 -626 -637 -683 -728 -786
60.5 63.3 63.3 65.1 67.9 73.7
10 25 50 100
150 200
-0.100
+
based on at least three determinations on the same aliquot. Table IV. Linearity of the Calibration Curves"
analyte diazepam indium lead
slope,b pAJppb 4.49 X 5.8 X 1.35 X
intercept, r A 0.02567 -0.05314 1.26363
sum of squared residuals 2.61 X 2.32 X 1.02 X
-0,800
-0.5
'500 ppb In3+ 20 ppm Hg2+in acetate buffer (pH 4.0). Deposition time is 15 s. bAverage peak current (oxidation) values
E(V) vs. Ag/AgCI
Figure 5. Thin-layer SW anodic stripping voltammogram for 100 ppb Pb2+, 20 ppm Hg2+,and pH 4.0 acetate buffer. Other parameters are given in Figure 3.
6.0
RMS error
i
0.01022 0.00963 0.0336
aBased on three values of each point. bExcept in the case of diazepam, where the slope is rA/ppm.
%
al. (13)that square wave amplitudes beyond 50 mV offer very little gain in sensitivity due to problems of charging currents caused by the high frequency of the square wave cycles. The increases in square wave amplitude also caused the peak potential to shift to a much more negative potential. The results of the optimization of the square wave amplitude are summarized in Table 111. Calibration plot data over the concentration range from 100 to 5000 ppb was obtained (Table IV). The plot is linear up M In3+) with a detection limit of 8 to 2000 ppb (1.74 X ppb (6.97 X M In3+; 2.1 pmol). Our calibration curve studies on indium solutions containing 20 ppm H$+ also show that successive in situ 60-s depositions and anodic scans (without removing the mercury film)cause a positive shift in the peak potential, E,,.The shift in peak potential is probably due to the increase in the mercury film thickness. Table V shows the results of successive depositions and anodic scans on one aliquot of a 500 ppb In3+ solution. Each deposition was commenced immediately after completion of the previous scan without disturbing the electrodes. Batley Florence reported similar results for their DPASV study of T1+ a t the GCE and the HMDE (27). Determination of Lead. Lead is an important pollutant in man's environment. Ib detection in blood is one of the most frequent determinations of a metal. Consequently, the determination of lead was selected as another analyte with which to test the utility of thin-layer SWSV. A typical voltammo-
8
t t
2.0
01
0
1
20
40
60
80
100
120
140
160
180
DEPOSITION TIME (sac.)
Flgure 6. Peak current (oxidation)of lead stripping peak as a function of deposition time: Deposition potential = -0.95 V vs. AgIAgCi; 100 ppb Pb2+, 20 ppm Hg2+, and pH 4 acetate buffer.
gram of Pb2+is shown in Figure 5. The preliminary data for the calibration plot was obtained without preelectrolyzing the acetate buffer solution. The data was obtained for standard solutions in the concentration range 100-10 000 ppb. Each value of current represents the average of three analyses. The plot of peak current vs. concentration was linear with a detection limit of ll ppb (5.31 x lo4 M Pb2+;1.59 pmol). Table IV shows the least-squares results for the calibration plot data. The effect of deposition time on the peak height of the SWSV stripping peak of lead was investigated. Figure 6 shows the peak current for deposition times varying from 15 to 180 s. The current increases rapidly for deposition times up to about 60 s after which the rate of current increase is negligible. The smaller increase in current for longer deposition times is due to diffusion into the cell along the edges of the electrode (28). On the basis of these data, it may be concluded that complete electrolysis of Pb2+ and other ions is achieved in about 60 s.
846
Anal. Chem. 1987, 59,846-850
LITERATURE CITED
Ullucci, P. A.; Cadoret, R.; Stasiowski, P. D.; Martin, H. F. J . Anal. roxicoi. 1978, 2 ( 2 ) ,33-38. Greenblatt, D. J. Clin. Chem. (Winsfon-Salem, N.C.) 1978, 2 4 , 1838-1841. Baselt, R . C.; Stewart, C. B.; Franch. S. J. J . Anal. Toxicol. 1977, 7 ( I ) , 10-13. Bailey, L. C. A m . J . Pharm. Educ. 1078, 4 2 , 63-66. Perchalski, R. J.; Wilder, 6. J. Anal. Chem. 1978, 50, 554-557. Wallace, J. E.; Harris, S. C.; Shimek, E. L.. Jr. Clin. Chem. (WinstonSalem, N.C.) 1980, 2 6 , 1905-1907. Aderjan, R.; Schmidt, G. 2.Rechtsmod. 1979, 83, 191-200. Bourne, I?. C.; Robinson, J. D.; Teale, J. D. Br. J . Pharmacol. 1978, 63, 371P. Meola. J. M.; Rosano, T. G.; Swift, T. Clin. Chem. (Winston-Salem, N.C.) 1981, 2 7 , 1254-1255. Doyle, M. J.; Halsall, H. B.;Heineman, W. R. Anal. Chem. 1982, 5 4 , 2318-2322. Batley, G. E.; Florence, T. M. J . Eiecfroanal. Chem. 1974, 5 5 , 23-43. McDuffie. B.; Anderson, L. B.; Reilley. C. N. Anal. Chem. 1986, 36, 883-890.
DeAngelis, T. P.; Heineman, W. R. Anal. Chem. 1978, 4 6 , 2262-2263. DeAngelis, T. P.; Bond, R. E.; Brooks, E. E.; Heineman, W. R . Anal. Chem. 1977, 4 9 , 1792-1797. Jarbawi, T. B.; Heineman, W. R.: Patriarche, G. J Anal. Chim. Acta 1981, 126, 57-64. Wise, J. A.; Heineman, W. R. Anal. Chim. Acta 1985, 172, 1-12. Christie, J. H.; Turner. J. A.; Osteryoung, R. A. Anal. Cham. 1977, 4 9 , 1899-1903. Turner, J. A.; Christie, J. H.; Vukovic, M.: Osteryoung, R A . Anal. Chem. 1977, 4 9 , 1904-1908. Ramaley, L.; Krause, M. S., Jr. Anal. Chem. 1989, 4 7 , 1362-1365. Krause, M. S., Jr.; Ramaiey, L. Anal. Chem. 1969, 4 7 , 1365-1369. Buchanan, E. B., Jr.; Soleta, D. D. Talanfa 1982, 2 9 , 207-211. Buchanan, E. B., Jr.; Soleta, D. D. Talanta 1983, 30, 459-464. Osteryoung. J. G.; Osteryoung, R . A. Anal. Chem. 1985, 5 7 , 101A110A. Wojciechowski, M.; Go, W.; Osteryoung, J. Anal. Chem. 1985, 5 7 , 155-158. Turner, J. A.; Eisner, U.; Osteryoung, R. A. Anal. Chim. Acta 1977, 9 0 , 25--34. Eilaithy, M. M.; Volke, J.; Manousak. 0 . Talanta 1977, 2 4 , 137-140. Rao, G. R.; Kanjilal, G.; Srivastava, C. M. R. Indian J . Pharm. Sci. 1980, 4 2 , 63-64. Foltz. R. I.. Quant. Mass Specfrom. Life Sci 1978. 2 , 39-62.
RECEIVED for review August 18,1986. Accepted November 25,1986. V.K. acknowledges financial support received in the form of a Faculty Opportunity Award supplement to W.R.H. Grant CHE 8217045 from the National Science Foundation.
Effect of Sample Stream Radius upon Light Scatter Distributions Generated with a Gaussian Beam Light Source in the Sheath Flow Cuvette Fahimeh Zarrin' and Norman J. Dovichi*2 Department of Chemistry, Universit.y o f Wyoming, Laramie, Wyomifig 82071
hydrodynamic chromatography (8),and in the study of gaseous samples (IO). In the sheath flow cuvette, a sample is injected as a thin stream into a flowing sheath stream usually consisting of pure solvent. Under laminar flow conditions, the sample stream retains its identify as a narrow stream flowing in the center of the sheath fluid. Adjustment of the relative volume flow rates allows adjustment of the sample stream radius over a large range (9). The combination of high sheath and slow sample stream flow rates produces sample streams as small as a few micrometers in radius; interaction with a tightly focused laser beam yields very small probed volumes. Since the sample stream is surrounded by pure solvent, there is no refractive index boundary surrounding the sample stream to generate light scatter. This is in contrast to conventional flow cuvettes which generate light scatter at the cuvette wall-sample stream interface. This scattered light can be a significant source of background signal in laser-induced fluorescence and light scatter applications. For example, extremely low detection limits have been produced by laser-induced fluorescence within the sheath flow cuvette by restricting the field of view of the detector to the sample stream (1-7). In typical applications of the sheath flow cuvette, light scatter or fluorescence is observed at the intersection region of a sample stream and focused light beam, usually a continuous wave (CW) laser operating in the TEM,, or Gaussian spatial mode. The h e r beam crosses the sample stream at right angles. Fluorescence and light scatter may be detected along an axis orthogonal to the laser beam and sample stream axes. Light scatter also may be detected at small angles to the laser beam. In this paper we will be concerned with light scatter signals. The results also should be valid for fluores-
The light scatter intensity observed In slngle-partlcle counters depends upon the trajectory of the particle through the llght beam; particles that traverse low Intensity portions of the light beam will generate lower llght scatter slgnals than particles that pass through more Intense portions of the llght beam. To produce narrow llght scatter dlstrlbutlons uslng the sheath flow cuvette and a Gaussian laser beam, it is necessary that the sample stream radius be much smaller than the laser beam spot sire. A numerlcal model shows that a monodlspersed particle suspension will generate a llght scatter distrlbutlon wlth 12.5% relatlve standard deviation if the sample stream radlus equals half the laser beam spot size. A 5 % relative standard deviation Is produced If the sample stream radius is one-fifth the laser beam spot slze.
The sheath flow cuvette is useful in the optical analysis of small-volume samples (1-10). Primarily developed by the biomedical community for flow cytometry analysis of individual biological cells (11-26), the sheath flow cuvette has attracted some attention in the analytical community. The justification for analytical interest is simple: the cuvette may be used to produce subpicoliter detection volumes (9)and very low background signals. Analytical applications of the cuvette include laser-induced fluorescence detection for liquid chromatography (11, flow injection analysis ( 2 , 3 ) ,and attogram amounts of neat solution (4-7), as a light scatter detector in 'Present address: D e p a r t m e n t of Chemistry Colorado State University, Fort Collins, CO 80523. Present address: D e p a r t m e n t of Chemistry, U n i v e r s i t y of Alberta. Edmonton. Alberta, T6G 2G2 Canada.
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0003-270O/87/0359-0846$01.50/0 1987 American Chemical Society
